Engineering kinases for dual thymidine and thymidylate kinase activity 公开

Abstract

Engineering kinases for dual thymidine and thymidylate kinase activity

By Yichen Liu
Thymidine kinase (TK) and thymidylate kinase (TMPK) are essential enzymes
that function in the salvage pathway for synthesis of thymidine triphosphate, a DNA
building block. Their importance also exists in activating nucleoside analogs (NAs), an
important category of anti-cancer and anti-virus drugs. Because NAs enter the cell as
prodrugs, they require three consecutive phosphorylation steps catalyzed by cellular
kinases in the salvage pathway to become biologically active and halt DNA synthesis.
Among the cellular kinases responsible for NA activation, the first two
phosphorylation steps usually present the bottleneck because of their poor activity
towards NA. Previous engineering efforts to alter and improve specificity of these
kinases have largely focused on the first step of phosphorylation catalyzed by
deoxyribonucleoside kinase (dNK). However, such approach bears the real risk of simply
shifting the bottleneck from the improved first to the second phosphorylation reaction. A
more promising strategy is to eliminate both bottleneck steps by creating a dual
functioning enzyme that can convert NAs directly to their corresponding diphosphate
form. In this dissertation, I developed a conditional auxotroph E. coli strain for the
selection of dual TK and TMPK activity. As the first selection system reported for
selecting two consecutive reactions, this auxotroph strain enables the evaluation of

combinatorial kinase libraries for TK and TMPK activities both independently and
concurrently. Employing the dual-function auxotroph strain, I have evaluated chimera
libraries of dNK from Drosophila melanogaster and TMPK from Thermotoga maritima
using the non-homologous recombination technique SCRATCHY in combination with
the computational method SCHEMA. Separately, more fundamental questions
concerning the mechanistic similarities and differences between these two enzymatic
reactions were investigated by site-directed mutagenesis and ITC studies of substrate
binding. Experiments were conducted on DmdNK and TmTMPK, which excusively
catalyze the first or second phosphorylation reaction, as well as the only know dual-
function TK from herpes simplex virus (HSV). The information extracted from these
experiments provides new insights regarding the dual function of HSV1-TK and will
guide future engineering experiments.

Table of Contents

Table of Contents
Chapter 1 General introduction. ............................................................................. 1

1.1 Nucleoside analog as anti-cancer and anti-virus treatment. .................................. 2
1.2 Problems in NA activation by cellular salvage pathway and gene therapy .......... 5
1.3 Deoxyribonucleoside kinase and nucleoside monophosphate kinase ................... 9
1.3.1 dNK family ................................................................................................... 9
1.3.2 NMPK family ............................................................................................. 14
1.3.3 Multifunctional enzymes ............................................................................ 19
1.4 Engineering multifunctional kinase and the limitation ....................................... 20
1.5 Aim and scope of the dissertation ....................................................................... 23
Chapter 2 Expression and characterization of thymidylate kinase from
Thermotoga maritima ................................................................................................ 24
2.1 Introduction. ......................................................................................................... 25
2.2 Results and discussion .......................................................................................... 30
2.2.1 Structure and active site of TmTMPK ........................................................ 30
2.2.2 TmTMPK gene isolation and protein purification ..................................... 34
2.2.3 TmTMPK is a highly thermostable thymidylate kinase ............................. 36
2.2.4 TmTMPK in vitro catalytic performance ................................................... 38
2.2.5 Isothermal Titration Calorimetry (ITC) of TmTMPK ................................ 40
2.3 Conclusion remarks ............................................................................................. 45
2.4 Materials and methods ........................................................................................ 46
2.4.1 Materials ..................................................................................................... 46
2.4.2 Isolation of TmTMPK gene ........................................................................ 46

About this Dissertation

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School	Laney Graduate School
Department	Chemistry
Degree	PhD
Submission	Dissertation
Language	English
Research Field	Chemistry, General
关键词	protein engineering thymidylate kinase thymidine kinase
Committee Chair / Thesis Advisor	Lutz, Stefan, Emory University
Committee Members	Conticello, Vincent, Emory University Edmondson, Dale E, Emory University

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